This work was supported by National Institutes of Health Grants K08 NS061039-01A2, The Program for Neurology Research &#38; Discovery, and The A. Alfred Taubman Medical Research Institute at the University of Michigan. This work used the Morphology and Image Analysis Core of the Michigan Diabetes Research Center, funded by National Institutes of Health Grant 5P90 DK-20572 from the National Institute of Diabetes and Digestive and Kidney Diseases. The authors would like to thank J. Robinson Singleton and A. Gordon Smith (University of Utah) for their generous donation of human skin samples.

Skin punch biopsies were obtained from subjects that were recruited from a large community-based primary care network at the University of Utah Diabetes Center (Salt Lake City, UT). This study was approved by the University of Michigan Institutional Review Board and complied with the tenets of the Declaration of Helsinki. Written informed consent was obtained from each subject prior to testing.
1. Fluorescence Immunohistochemistry
<ol>
	<li>Prepare punch biopsies for intraepidermal nerve fiber immunohistochemistry:
	<ol>
		<li>Perform 3 mm skin biopsies by medical staff and place the whole biopsy in 1.5 mL Zamboni&#39;s fixative solution (2% paraformaldehyde, 0.3% saturated picric acid in phosphate buffered saline (PBS), pH 7.4) at 4 &#176;C overnight.</li>
		<li>Rinse samples with a solution of 30% sucrose in PBS at 4 &#176;C for 16-24 h or until the sample sinks.</li>
		<li>Embed samples in optimal cutting temperature compound (OCT) using a cryomold. Place the whole 3 mm biopsy with the epidermis facing down in the mold and fill the mold with approximately 2 mL of OCT. Freeze the mold on crushed dry ice. Store at -80 &#176;C until ready to use.</li>
		<li>Cut 50 &#181;m thick cross sections using a cryostat and store in individual wells of a 96-well plate using 180 &#181;L antifreeze storage solution per well (30% ethylene glycol, 30% glycerol in PBS). The following directions are for 8 wells of a 96 well plate. Stain sections from each biopsy site that are 200 - 300 &#181;m away from each other.</li>
	</ol>
	</li>
</ol>
DAY 1:
<ol start="2">
	<li>Quench non-specific labeling of the stratum corneum:
	<ol>
		<li>Label 96-well plate as shown in Figure 1.</li>
		<li>Pipet 150 &#181;L of stock signal enhancer solution to reduce nonspecific binding of secondary antibodies34,35 into each well. Transfer sections into signal enhancer solution using an inoculating loop. 
		NOTE: Take care while working with the tissue to avoid damaging or tearing the tissue sections. Keep in signal enhancer solution for 30 minutes at room temperature on a flat rocker.</li>
		<li>Prepare rinse wells in rows 2 and 3 of the 96-well plate by adding 150 &#181;L of 1x phosphate buffered saline (PBS) into each well.</li>
		<li>Carefully transfer sections into row 2 and rinse in 1x PBS for 10 min at room temperature.</li>
		<li>Rinse a second time in 1x PBS for 10 min at room temperature (row 3).</li>
	</ol>
	</li>
	<li>Prepare 5% bovine serum albumin (BSA) blocking solution:
	<ol>
		<li>Prepare 5% blocking solution that contains 5% BSA and 0.3% Triton X 100 (TX-100) in 0.1 M PBS (see Table 1) while sections are incubating in signal enhancer solution. BSA does not go into solution easily. Vortex solution until BSA completely dissolves.</li>
		<li>Prepare blocking wells in row 4 of the 96-well plate by adding 150 &#181;L of 5% BSA blocking solution into each well.</li>
		<li>Transfer sections into individual wells of 5% BSA blocking solution and incubate sections in 5% BSA blocking solution for 1 - 2 h at room temperature on a flat rocker.</li>
	</ol>
	</li>
	<li>Prepare 1% BSA rinsing solution and dilution of primary antibodies:
	<ol>
		<li>Prepare 1% rinsing solution that contains 1% BSA and 0.3% TX-100 in 0.1 M PBS (see Table 2) while sections are incubating in 5% BSA blocking solution. BSA does not go into solution easily. Vortex solution until BSA completely dissolves.</li>
		<li>Dilute primary antibodies in 1% BSA rinsing solution while sections are incubating in 5% BSA blocking solution.
		<ol>
			<li>Make 1,500 &#181;L of primary antibody solution and add to each well in row 5.</li>
			<li>Dilute the primary antibodies: Use the nerve-specific label, rabbit polyclonal anti-protein gene product 9.5 (PGP9.5) at 1:1,000. Use the mitochondria-specific label, mouse monoclonal anti-pyruvate dehydrogenase E2/E3bp antibody (PDH) at 1:100 in 1% BSA rinsing solution.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Prepare primary antibody:
	<ol>
		<li>Pipet 150 &#181;L of primary antibody into each well in row 5 of the 96-well plate.</li>
		<li>Using the loop tool, transfer sections from the blocking solution (row 4) into the row 5 containing the primary antibody.</li>
		<li>Wrap the plate tightly with parafilm to keep it from drying out.</li>
		<li>Place samples on flat rocker at room temperature for 1 h, and then incubate samples at 4 &#186;C on a flat rocker overnight.</li>
	</ol>
	</li>
</ol>
DAY 2:
<ol start="6">
	<li>Rinse samples:
	<ol>
		<li>Prepare rinse wells in rows 6, 7 and 8 of the 96-well plate by adding 150 &#181;L of 1% BSA rinsing solution into each well.</li>
		<li>Transfer sections into first 1% BSA rinsing solution (row 6) and incubate for 1 h at room temperature. Repeat rinses twice more by incubating sections in 1% BSA rinsing solution in rows 7 and 8 for 1 h each at room temperature.</li>
	</ol>
	</li>
	<li>Dilute secondary antibodies in 1% BSA rinsing solution:
	<ol>
		<li>Make 1,500 &#181;L of secondary antibody solution while sections are incubating in the last rinse of 1% BSA rinsing solution (row 8).</li>
		<li>Dilute secondary antibodies: for PGP9.5 (green-fluorescent conjugated goat anti-rabbit antibody, 1:1000), for PDH (red-fluorescent conjugated goat anti-mouse, 1:1,000) in 1% BSA rinsing solution.</li>
	</ol>
	</li>
	<li>Prepare secondary antibody:
	<ol>
		<li>Pipet 150 &#181;L of secondary antibody into row 9 of the 96-well plate.</li>
		<li>Gently transfer the sections from 1% BSA rinsing solution (row 8) into secondary antibody wells (row 9).</li>
		<li>Using parafilm, wrap the plate tightly to keep it from drying out. Cover with aluminum foil. Place samples on flat rocker at room temperature for 1 h, and then incubate samples at 4 &#730;C on a flat rocker overnight.</li>
	</ol>
	</li>
</ol>
DAY 3:
<ol start="9">
	<li>Prepare clean 1x PBS by filtering through a 0.22 &#181;m filter:
	<ol>
		<li>Pipet 150 &#181;L of filtered 1x PBS into row 10, 11, and 12.</li>
		<li>Transfer samples to row 10 and rinse in 1x PBS for 1 h at room temperature. Cover the 96-well plate with aluminum foil and place on a flat rocker during rinse. Repeat filtered 1x PBS rinse two more times for 1 h each at room temperature in rows 11 and 12.</li>
	</ol>
	</li>
	<li>Prepare microscope slides for mounting tissue sections:
	<ol>
		<li>Place 50 &#181;L of filtered 1x PBS on a slide.</li>
		<li>Transfer section from 1x PBS (row 12) into the 50 &#181;L drop. Carefully position the section in the drop of PBS by unfolding the tissue and gently flattening it onto the glass slide. Remove excess PBS with a glass pipette to avoid diluting the mounting reagent. Do not touch sections with the glass bulb.</li>
		<li>Pipet 1 - 2 drops of mounting reagent containing DAPI directly on top of the section on the microscope slide using care to not disturb the orientation of the section. Gently place a 50 mm x 24 mm #1.5 microscope glass coverslip over the section.</li>
		<li>Clear any air bubbles that formed while placing the coverslip and wipe excess liquid off the edges of the coverslip. Prepare a new slide and repeat the mounting process for each section.</li>
		<li>Cure/dry the mounting media by placing the slides in the dark overnight at room temperature. Transfer the slides to 4 &#176;C for short-term (1 - 2 weeks) or -20 &#176;C for long-term storage (greater than 2 weeks). 
		NOTE: Negative controls omitted primary antibodies for PGP9.5 or PDH in step 1.4.2.2 and displayed no distinguishable labeling of nerves or mitochondria in the epidermis. Positive controls were performed for the PDH antibody to prove it labels all mitochondria (data not shown). Positive controls were performed on cultured primary mouse dorsal root ganglion neurons that were transduced with a baculovirus to label mitochondria with a Green Fluorescent Protein (GFP) signal and then fixed and stained with the PDH antibody and red fluorescent secondary antibody. All mitochondria that were expressing GFP were co-labeled with the red label for PDH immunohistochemistry (data not shown).</li>
	</ol>
	</li>
</ol>
2. Confocal Imaging
<ol>
	<li>Perform confocal imaging:
	<ol>
		<li>Collect images using a laser scanning confocal microscope with a 40X oil-immersion (1.25 numerical aperture (N.A.)) objective on an inverted microscope.
		<ol>
			<li>At each focal plane sequentially acquire fluorescent signals: 
			Nuclei: excitation &#955; = 405 nm, spectral emission filter &#955; = 420 - 480 nm 
			Nerve fibers: excitation &#955;= 488 nm, spectral emission filter &#955; = 505 - 560 nm 
			Mitochondria: excitation &#955; = 543 nm, spectral emission filter &#955;= 606 - 670 nm</li>
		</ol>
		</li>
		<li>Enter the following scan parameters into the microscope software: scan rate of 600 Hz with 2 frame averaging and zoom of 2.2; 12-bit intensity resolution (4096 gray levels).
		<ol>
			<li>Set the microscope software for optimized lateral resolution (scan resolution = 1,024 x 1,024) and axial resolution/optical sectioning (confocal aperture = 1 airy unit (AU) with z-step size of 210 nm). 
			NOTE: The resulting XYZ resolution is 172.2 nm x 172.2 nm x 210 nm with an image size of 176.1 &#181;m x 176.1 &#181;m x 30-50 &#181;m.</li>
		</ol>
		</li>
		<li>Activate a live scan for the nerve signal (green-fluorescence) and adjust the z-focus control to find and set the upper and lower focal planes in the microscope software that encompass the nerve signal within the tissue section. The total z-range is typically 30-50 &#181;m for a 50 &#181;m tissue section.</li>
		<li>Rotate the scan field with the microscope software during a live scan so that the epidermis is horizontally or vertically positioned in the image.</li>
		<li>Scan each signal separately and adjust the detector (photomultiplier tube, PMT) voltage and offset to minimize/remove any over and under saturated pixels. 
		NOTE: Scan times with the above parameters take approximately 20 - 40 min depending on the number of z slices.</li>
	</ol>
	</li>
</ol>
3. 3D Visualization and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers
<ol>
	<li>Isolate the 3D epidermis:
	<ol>
		<li>Duplicate the original image and use the maximum intensity projection (extended focus view) of the image to identify and isolate the epidermis.</li>
		<li>Use a region of interest tool to trace along the upper and lower edges of the epidermis to remove unwanted areas such as the stratum corneum and dermis that are absent of intraepidermal nerve fibers. Crop to this selection.</li>
	</ol>
	</li>
	<li>Use deconvolution on the nerve and mitochondrial fluorescent signals: 
	NOTE: Deconvolution helps to restore the integrity of the fluorescent signals. The restoration used in this protocol is called blind deconvolution because it uses the fluorescent signals in the images to determine how much the signals spread from their original source (point spread function). The process improves signal resolution by reassigning the signal spread back to its origin location.
	<ol>
		<li>Calculate a point spread function (PSF) for the green fluorescent nerve signal (green-fluorescence) with the following parameters:
		<ol>
			<li>Set calculated PSF to confocal. Set medium refractive index to 1.515 and numerical aperture to 1.25. Set detector pinhole to 1 Airy unit (A.U.). Set laser excitation wavelength to 488 nm and emission wavelength to 515 nm.</li>
		</ol>
		</li>
		<li>Calculate a PSF for the red fluorescent mitochondrial signal (red-fluorescence) with the following parameters:
		<ol>
			<li>Set calculated PSF to confocal. Set medium refractive index to 1.515 and numerical aperture to 1.25. Set detector pinhole to 1 AU. Set laser excitation wavelength to 543 nm and emission wavelength to 617 nm.</li>
		</ol>
		</li>
		<li>Optimize the nerve and mitochondria fluorescent signals by deconvolution using the corresponding PSFs listed above and iterative restoration feature set at 100% confidence and an iteration limit of 10 cycles.</li>
	</ol>
	</li>
	<li>Create nerve-specific surfaces:
	<ol>
		<li>Use the &#34;create surface&#34; tool to make a solid surface of the nerves from the deconvolved green- fluorescent secondary labeling of the PGP9.5 identified nerves.</li>
		<li>Uncheck the &#34;smooth&#34; feature and use the absolute intensity feature to set the threshold for the nerve signal since it is significantly brighter than background fluorescence.</li>
		<li>Use the absolute intensity feature to set the threshold for the nerve signal since it is significantly brighter than background fluorescence. Set threshold value low enough to accurately identify the nerves.</li>
		<li>Filter out small, non-nerve surfaces based on size. 
		NOTE: If necessary, manually edit out additional non-nerve surfaces within the &#34;Edit&#34; tab by holding the CONTROL key to highlight multiple objects and then deleting them with the Delete key.</li>
	</ol>
	</li>
	<li>Isolate nerve-specific fluorescent mitochondrial signal:
	<ol>
		<li>Select the Edit tab of the nerve surface to view the &#34;Mask Properties&#34; feature. The nerve surface created in steps 3.3 is used to isolate mitochondria within those nerves away from mitochondrial signals associated with the keratinocytes.</li>
		<li>As the &#34;Mask All&#39; button opens a &#34;Mask Channel&#34; window, choose the deconvolved red-fluorescent signal from the pull down menu under &#34;Channel Selection&#34; for the mitochondrial signal.</li>
		<li>Click in the box to put a check mark in the &#34;duplicate channel before applying mask&#34; option.</li>
		<li>Click on the radio button in front of the &#34;Constant inside/outside&#34; option of the Mask Settings and click in the box to put a check mark in the &#34;Set voxel outside surface to&#34; option and type in 0.00 for the value. Click OK button to create the new channel that represents mitochondrial signals within the nerve surface.</li>
	</ol>
	</li>
	<li>Create mitochondria-specific surfaces:
	<ol>
		<li>Use the &#34;create surface&#34; tool to make a solid surface of the mitochondria from the newly created fluorescent channel of the nerve-specific mitochondrial signals from steps 3.4.</li>
		<li>Uncheck the &#34;smooth&#34; feature and select the &#34;background subtraction&#34; feature to set the threshold. This feature uses local contrast around the mitochondrial signal to identify mitochondria from background.</li>
		<li>Set threshold value low enough to accurately identify mitochondria. In this example the lower threshold was set at 2,000 for a 16-bit (65,536) scale.</li>
		<li>Filter mitochondrial surfaces based on size. In this example the voxel limit was set to 1.0 voxels, which is the lowest limit possible. If necessary, manually edit out non-mitochondria surfaces within the &#34;Edit&#34; tab by holding the CONTROL key to select multiple objects and then deleting them with the Delete key. 
		NOTE: Occasionally, the software will create surfaces that are not associated with a distinguishable fluorescent mitochondrial signal. In these cases, it is possible to remove them with the &#34;Edit&#34; tab.</li>
	</ol>
	</li>
	<li>Export and calculate morphometric values for analysis:
	<ol>
		<li>Export values for nerve and mitochondrial surfaces from the &#34;Statistics&#34; tab for further analysis with electronic spreadsheet software.</li>
		<li>Export the volume values for both the nerve and mitochondrial surfaces.</li>
		<li>Count the number of individual nerve fibers present in each image and record the value in the electronic spreadsheet.</li>
		<li>For each image, calculate the following potential values for analysis in the electronic spreadsheet:
		<ol>
			<li>Sum the volumes for all nerve surfaces.</li>
			<li>Filter out nerve-specific mitochondrial surfaces below a volume of less than 0.02 &#181;m3 and bin the surfaces by size. For example, use bins such as 0.02 - 0.04, 0.04 - 0.08, 0.08 - 0.16, 0.16 - 0.32, 0.32 - 0.64, 0.64 - 1.28, 1.28 - 2.56, greater than 2.56 &#181;m3. 
			NOTE: The lower limit of mitochondrial volume was set to 0.02 &#181;m3 based on previously published volumes of mitonchodria36,37,38.</li>
			<li>Count the number of nerve-specific mitochondrial surfaces within each bin and the total number of surfaces over the bins (0.02 - greater than 2.56 &#181;m3).</li>
			<li>Calculate the proportion of nerve-specific mitochondrial surfaces in each bin. Use the count per bin divided by the total number of mitochondria surfaces.</li>
			<li>Sum the volumes for all nerve-specific mitochondrial surfaces.</li>
			<li>Calculate the proportion of nerve with mitochondrial signal by dividing the total nerve surface volume by the sum of all mitochondrial surface volumes.</li>
			<li>Calculate the number of mitochondria per nerve volume by dividing the total nerve surface volume by the count of all mitochondrial surfaces.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

No conflicts of interest to declare.

This protocol is designed to isolate, quantify and analyze the size and distribution of nerve-specific mitochondria within IENFs in 3D from human skin biopsies. There are several critical steps in the protocol. The free-floating fluorescence immunohistochemistry is designed to stain and analyze multiple signals in each sample, providing a more versatile methodology for explorative research44,45. This procedure allows for penetration of the antibodies into the tis...

Mitochondria serve vital cellular functions that include producing cell energy, buffering calcium, and regulating necrotic and apoptotic cell death1,2,3. The nervous system has a high metabolic rate compared to the body4 suggesting that neurons generate a high degree of cellular energy in the form of adenosine triphosphate (ATP) through mitochondrial respiration. A lot of evidence documents that neuronal functions are dependent on ATP5, especially at the synapses6. Therefore, the distribution of mitochondria within neurons is important.
Over the last 10 years a lot of information has shown that the trafficking and docking of neuronal mitochondria is highly regulated. Motor proteins are involved in distributing mitochondria to specific cellular compartments throughout the neuron. Trafficking of mitochondria is particularly important because neurons project axons and dendrites far away from the soma. Kinesin motor proteins primarily direct anterograde (away from the soma) trafficking of mitochondria along microtubules while dynein motor proteins direct retrograde (toward the soma) motility7,8,9,10. There are cellular signals such a mitochondrial membrane potential and impulse conduction that influence the presence and direction of mitochondrial trafficking11,12,13.
In addition to transporting mitochondria, there are specialized proteins to localize mitochondria to specific cellular compartments that have high energy demands, such as nodes of Ranvier and synapses8,14,17. In fact, the majority of mitochondria within axons are non-motile9,13,18. Specialized proteins like syntaphilin anchor mitochondria to microtubules along axons while other proteins anchor mitochondria to the actin cytoskeleton19-21. Growth factors and ions such as calcium have been reported to support the cessation of mitochondria movement to localize them to regions where they are needed21,22,23.
Taken together, the trafficking and docking of mitochondria are vital for proper function of neurons. In support of this, disruption in mitochondrial trafficking has been associated with several neurological conditions including Alzheimer&#39;s disease, amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, Huntington&#39;s disease, hereditary spastic paraparesis, and optic atrophy15,24,25,26,27. Recent studies have focused on mitochondrial dysfunction and pathology as a potential mechanism for diabetic neuropathy, the sensory loss associated with diabetes28,29,30,31,32,33. The hypothesis is that diabetes alters the distribution of mitochondria within the sensory projections of cutaneous nerve ending. Therefore, a technique was developed to visualize and quantify mitochondria within the intraepidermal nerve fibers (IENFs), the distal tips of dorsal root ganglion sensory afferents. The technique combines fluorescence immunohistochemistry of specific mitochondrial and nerve fiber labels with confocal microscopy z-series acquisition of signals with powerful 3D image analysis software to measure the distribution of nerve-specific mitochondria from human cutaneous punch biopsies to achieve this goal.

<table><tbody><tr><td>2% Zamboni&#039;s Fixative</td><td>Newcomer Supply, Middleton, WI&amp;nbsp;</td><td>1459A</td><td>2% paraformaldehyde, 0.2% saturated picric acid in phosphate buffered saline (PBS), pH 7.4</td></tr><tr><td>10x Phosphate Buffered Saline (PBS)&amp;nbsp;</td><td>Fisher Scientific, Pittsburgh, PA</td><td>BP399-4</td><td>To make up 1x PBS</td></tr><tr><td>Image-iT FX Signal Enhancer</td><td>ThermoFisher Scientific, Waltham, Massachusetts</td><td>I36933</td><td>enhances Alexa Fluor dye signals by reducing nonspecific binding</td></tr><tr><td>Anti-Protein Gene Product 9.5 Antibody (Rabbit Polyclonal)</td><td>Proteintech Group Inc. Rosemont, IL</td><td>14730-1-AP</td><td>abbreviated as PGP9.5, replaces discontinued AbD Serotec (Cat. No. 7863-0504) antibody</td></tr><tr><td>Anti-Pyruvate Dehydrogenase E2/E3bp Antibody (Mouse Monoclonal)</td><td>abcam, Cambridge, MA</td><td>ab110333</td><td>abbreviated as PDH</td></tr><tr><td>Goat anti-mouse Secondary antibody Alexa Fluor 594 conjugate</td><td>ThermoFisher Scientific, Waltham, Massachusetts</td><td>A-11034</td><td>red-fluorescent conjugated secondaryantibody</td></tr><tr><td>Goat anti-rabbit Secondary antibody Alexa Fluor 488 conjugate</td><td>ThermoFisher Scientific, Waltham, Massachusetts</td><td>A-11032</td><td>green-fluorescent conjugated secondaryantibody</td></tr><tr><td>Albumin, from Bovine Serum</td><td>Sigma-Aldrich, St. Louis, MO</td><td>A7906-100</td><td>abbreviated as BSA</td></tr><tr><td>Triton X- 100</td><td>Sigma-Aldrich, St. Louis, MO</td><td>T9284</td><td>abbreviated as TX-100</td></tr><tr><td>0.22 &amp;micro;m Filter</td><td>EMD Millipore, Billerica&lt;br/&gt; MA</td><td>MILLEX GP SLGP 033NS</td><td>0.22 &amp;micro;m Millipore filter</td></tr><tr><td>Parafilm M</td><td>Fisher Scientific, Pittsburgh, PA</td><td>13-374-10</td><td>Curwood Wisconsin LLC Parafilm M (PM-996)</td></tr><tr><td>Non-calibrated Loop</td><td>Fisher Scientific, Pittsburgh, PA</td><td>22-032092</td><td>inoculating Loop by Decon LeLoop (MP 199-25)</td></tr><tr><td>96-well Assay Plate</td><td>Corning Incorporated, Corning, NY</td><td>3603</td><td>96-well flat bottom plate</td></tr><tr><td>Prolong Gold antifade reagent with DAPI</td><td>ThermoFisher Scientific, Waltham, Massachusetts</td><td>P-36931</td><td>DAPI staining of nuclei</td></tr><tr><td>Microscope Cover Glass 50 x 24 mm</td><td>Fisher Scientific, Pittsburgh, PA</td><td>12-544E</td><td>Coverslips</td></tr><tr><td>Superfrost Plus Microscope Slides</td><td>Fisher Scientific, Pittsburgh, PA</td><td>12-550-15</td><td>Microscope Slides</td></tr><tr><td>Leica SP5 Laser Scanning Confocal Microscope</td><td>Leica Microsystems, Buffalo Grove, IL</td><td>SP5</td><td>Confocal Microscope</td></tr><tr><td>Volocity x64 Software&amp;nbsp;</td><td>Perkin Elmer, Waltham , MA</td><td>version 4.4.0</td><td>Volocity software is used for Steps 3.1 and 3.2 in the protocol for image processing</td></tr><tr><td>Imaris x64 3 Dimensional Analysis Software</td><td>Bitplane, Concord, MA</td><td>version 7.7.1</td><td>Imaris software is used for Steps 3.3 through 3.5 in the protocol for image analysis</td></tr><tr><td>Excel</td><td>Microsoft, Redmond, WA</td><td>version Office 2013</td><td>Excel spreadsheet software is used for Step 3.6 in the protocol to summarize morphometric features</td></tr><tr><td>Optimum Cutting Temperature Compound</td><td>Sakura Finetek USA, Inc., Torrance, CA</td><td>4583</td><td>abbreviated as OCT</td></tr><tr><td>Leica Cryostat</td><td>Leica Biosystems, Buffalo Grove, IL</td><td>CM1850</td><td>Cryostat for cutting 50 &amp;micro;m sections</td></tr><tr><td>CellLight Mitochondria-GFP, BacMam 2.0</td><td>ThermoFisher Scientific, Waltham, Massachusetts</td><td>C10600</td><td>Used as a postive control to label mitochondria with a green fluorescent signal</td></tr></tbody></table>

three-dimensional imaging and analysis of mitochondria within human intraepidermal nerve fibers

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

Visualization and quantification of mitochondria within human IENFs
Fluorescence immunohistochemistry allows for the simultaneous labeling of multiple signals within human skin biopsies to visualize nerves, mitochondria, and nuclei. A 96-well plate is a convenient way to organize the steps in the immunohistochemistry procedure. Figure 1 shows that this configuration accounts for up ...

Watch this Scientific Journal Video about Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers at JoVE.com

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...

three-dimensional-imaging-analysis-mitochondria-within-human

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

10.2K Views.  University of Michigan Medical School. This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal. 

Video: Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. At present, it is common practice to collect 3 mm skin biopsies from the distal leg (DL) and the proximal thigh (PT) for the evaluation of length-dependent polyneuropathies 3. However, due to the multidirectional nature of IENFs, it is challenging to examine overlapping nerve structures through the analysis of two-dimensional (2D) imaging. Alternatively, three-dimensional (3D) imaging could provide a better solution for this dilemma.
In the current report, we present methods for applying 3D imaging to study painful neuropathy (PN). In order to identify IENFs, skin samples are processed for immunofluorescent analysis of protein gene product 9.5 (PGP), a pan neuronal marker. At present, it is standard practice to diagnose small fiber neuropathies using IENFD determined by PGP immunohistochemistry using brightfield microscopy 4. In the current study, we applied double immunofluorescent analysis to identify total IENFD, using PGP, and nociceptive IENF, through the use of antibodies that recognize tropomyosin-receptor-kinase A (Trk A), the high affinity receptor for nerve growth factor 5. The advantages of co-staining IENF with PGP and Trk A antibodies benefits the study of PN by clearly staining PGP-positive, nociceptive fibers. These fluorescent signals can be quantified to determine nociceptive IENFD and morphological changes of IENF associated with PN. The fluorescent images are acquired by confocal microscopy and processed for 3D analysis. 3D-imaging provides rotational abilities to further analyze morphological changes associated with PN. Taken together, fluorescent co-staining, confocal imaging, and 3D analysis clearly benefit the study of PN.

In order to study the changes of nociceptive intraepidermal nerve fibers (IENFs) in painful neuropathies (PN), we developed protocols that could directly examine three-dimensional morphological changes observed in nociceptive IENFs. Three-dimensional analysis of IENFs has the potential to evaluate the morphological changes of IENF in PN.

A punch biopsy of the skin is commonly used to quantify intraepidermal nerve fiber densities (IENFD) for the diagnosis of peripheral polyneuropathy 1,2. ...

Medicine

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their close-to-surface positioning facilitates microscopic imaging of skin nerve endings in living intact animal. Using multiphoton microscopy, it is possible to obtain fine images overcoming the problem of strong light scattering of the skin tissue. Reporter transgenic mice that express EYFP under the control of Thy-1 promoter in neurons (including periphery sensory neurons) are well suited for the longitudinal studies of individual nerve endings over extended periods of time up to several months or even life-long. Furthermore, using the same femtosecond laser as for the imaging, it is possible to produce highly selective lesions of nerve fibers for the studies of the nerve fiber restructuring. Here, we present a simple and reliable protocol for longitudinal multiphoton in vivo imaging and laser-based microsurgery on mouse skin nerve endings.

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is presented.&#160;

Nerve endings in skin are involved in physiological processes such as sensing1 as well as in pathological processes such as neuropathic pain2. Their ...

3D Technique to Visualize Mitochondrial Cristae in Pancreatic Cancer Cells

A Three-Dimensional Technique for the Visualization of Mitochondrial Ultrastructural Changes in Pancreatic Cancer Cells

Understanding the dynamic features of the cell organelle ultrastructure, which is not only rich in unknown information but also sophisticated from a three-dimensional (3D) perspective, is critical for mechanistic studies. Electron microscopy (EM) offers good imaging depth and allows for the reconstruction of high-resolution image stacks to investigate the ultrastructural morphology of cellular organelles even at the nanometer scale; therefore, 3D reconstruction is gaining importance due to its incomparable advantages. Scanning electron microscopy (SEM) provides a high-throughput image acquisition technology that allows for reconstructing large structures in 3D from the same region of interest in consecutive slices. Therefore, the application of SEM in large-scale 3D reconstruction to restore the true 3D ultrastructure of organelles is becoming increasingly common. In this protocol, we suggest a combination of serial ultrathin section and 3D reconstruction techniques to study mitochondrial cristae in pancreatic cancer cells. The details of how these techniques are performed are described in this protocol in a step-by-step manner, including the osmium-thiocarbohydrazide-osmium (OTO) method, the serial ultrathin section imaging, and the visualization display.

Author Spotlight: A Three-Dimensional Technique for the Visualization of Mitochondrial Ultrastructural Changes in Pancreatic Cancer Cells  

This protocol describes how to reconstruct mitochondrial cristae to achieve 3D imaging with high accuracy, high resolution, and high throughput.

Understanding the dynamic features of the cell organelle ultrastructure, which is not only rich in unknown information but also sophisticated from a ...

Cancer Research

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis muscle of the garter snake. Raw data comprises time-lapse sequences of 3D z-stacks. Each stack contains 4-20 images acquired with epifluorescence optics at focal planes separated by 400-1,500 nm. Steps in the acquisition of image stacks, such as adjustment of focus, switching of excitation wavelengths, and operation of the digital camera, are automated as much as possible to maximize image rate and minimize tissue damage from light exposure. After acquisition, a set of image stacks is deconvolved to improve spatial resolution, converted to the desired 3D format, and used to create a 4D "movie" that is suitable for variety of computer-based analyses, depending upon the experimental data sought. One application is study of the dynamic behavior of two classes of endosomes found in nerve terminals-macroendosomes (MEs) and acidic endosomes (AEs)-whose sizes (200-800 nm for both types) are at or near the diffraction limit. Access to 3D information at each time point provides several advantages over conventional time-lapse imaging. In particular, size and velocity of movement of structures can be quantified over time without loss of sharp focus. Examples of data from 4D imaging reveal that MEs approach the plasma membrane and disappear, suggesting that they are exocytosed rather than simply moving vertically away from a single plane of focus. Also revealed is putative fusion of MEs and AEs, by visualization of overlap between the two dye-containing structures as viewed in each three orthogonal projections.

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals. Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, tri-carboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) pathways to produce cellular energy in the form of adenosine triphosphate (ATP). Impairment of mitochondrial ATP production causes mitochondrial disorders, which present clinically with prominent neurological and myopathic symptoms. Mitochondrial defects are also present in neurodevelopmental disorders (e.g. autism spectrum disorder) and neurodegenerative disorders (e.g. amyotrophic lateral sclerosis, Alzheimer&#39;s and Parkinson&#39;s diseases). Thus, there is an increased interest in the field for performing 3D analysis of mitochondrial morphology, structure and distribution under both healthy and disease states. The brain mitochondrial morphology is extremely diverse, with some mitochondria especially those in the synaptic region being in the range of &#60;200 nm diameter, which is below the resolution limit of traditional light microscopy. Expressing a mitochondrially-targeted green fluorescent protein (GFP) in the brain significantly enhances the organellar detection by confocal microscopy. However, it does not overcome the constraints on the sensitivity of detection of relatively small sized mitochondria without oversaturating the images of large sized mitochondria. While serial transmission electron microscopy has been successfully used to characterize mitochondria at the neuronal synapse, this technique is extremely time-consuming especially when comparing multiple samples. The serial block-face scanning electron microscopy (SBFSEM) technique involves an automated process of sectioning, imaging blocks of tissue and data acquisition. Here, we provide a protocol to perform SBFSEM of a defined region from rodent brain to rapidly reconstruct and visualize mitochondrial morphology. This technique could also be used to provide accurate information on mitochondrial number, volume, size and distribution in a defined brain region. Since the obtained image resolution is high (typically under 10 nm) any gross mitochondrial morphological defects may also be detected.

Mitochondrial visualization and analysis from mammalian brain tissue is a challenging task. Here, we describe how three dimensional (3D) reconstruction analysis from the serial block-face scanning electron microscopy (SBFSEM) can be used to gain insights on the morphological and volumetric analysis of this critical energy generating organelle.

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, ...

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human neurons, which may contribute to neurodegeneration in various disease states. Although it is challenging to examine mitochondrial dynamics in live human nerves, such paradigms are critical for studying the role of mitochondria in neurodegeneration. Described here is a protocol for analyzing mitochondrial transport and mitochondrial morphology in forebrain neuron axons derived from human induced pluripotent stem cells (iPSCs). The iPSCs are differentiated into telencephalic glutamatergic neurons using well-established methods. Mitochondria of the neurons are stained with MitoTracker CMXRos, and mitochondrial movement within the axons are captured using a live-cell imaging microscope equipped with an incubator for cell culture. Time-lapse images are analyzed using software with &#34;MultiKymograph&#34;, &#34;Bioformat importer&#34;, and &#34;Macros&#34; plugins. Kymographs of mitochondrial transport are generated, and average mitochondrial velocity in the anterograde and retrograde directions is read from the kymograph. Regarding mitochondrial morphology analysis, mitochondrial length, area, and aspect ratio are obtained using the ImageJ. In summary, this protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology to facilitate studies of neurodegenerative diseases.

Impaired mitochondrial transport and morphology are involved in various neurodegenerative diseases. The presented protocol uses induced pluripotent stem cell-derived forebrain neurons to assess mitochondrial transport and morphology in hereditary spastic paraplegia. This protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology, which will facilitate the study of neurodegenerative disease.

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human ...

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study of this movement in situ was only possible using radioisotopic pulse-labeling, which permits analysis of axonal transport in whole nerves with a temporal resolution of days and a spatial resolution of millimeters. To study neurofilament transport in situ with higher temporal and spatial resolution, we developed a hThy1-paGFP-NFM transgenic mouse that expresses neurofilament protein M tagged with photoactivatable GFP in neurons. Here we describe fluorescence photoactivation pulse-escape and pulse-spread methods to analyze neurofilament transport in single myelinated axons of tibial nerves from these mice ex vivo. Isolated nerve segments are maintained on the microscope stage by perfusion with oxygenated saline and imaged by spinning disk confocal fluorescence microscopy. Violet light is used to activate the fluorescence in a short axonal window. The fluorescence in the activated and flanking regions is analyzed over time, permitting the study of neurofilament transport with temporal and spatial resolution on the order of minutes&#160;and microns, respectively. Mathematical modeling can be used to extract kinetic parameters of neurofilament transport including the velocity, directional bias and pausing behavior from the resulting data. The pulse-escape and pulse-spread methods can also be adapted to visualize neurofilament transport in other nerves. With the development of additional transgenic mice, these methods could also be used to image and analyze the axonal transport of other cytoskeletal and cytosolic proteins in axons.

We describe fluorescence photoactivation methods to analyze the axonal transport of neurofilaments in single myelinated axons of peripheral nerves from transgenic mice that express a photoactivatable neurofilament protein.

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study ...

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal transport have devastating consequences for individual neurons and their networks, and contribute to a plethora of neurological disorders. As many of these conditions involve both cell autonomous and non-autonomous mechanisms, and often display a spectrum of pathology across neuronal subtypes, methods to accurately identify and analyze neuronal subsets are imperative.
This paper details protocols to assess in vivo axonal transport of signaling endosomes and mitochondria in sciatic nerves of anesthetized mice. Stepwise instructions are provided to 1) distinguish motor from sensory neurons in vivo, in situ, and ex vivo by using mice that selectively express fluorescent proteins within cholinergic motor neurons; and 2) separately or concurrently assess in vivo axonal transport of signaling endosomes and mitochondria. These complementary intravital approaches facilitate the simultaneous imaging of different cargoes in distinct peripheral nerve axons to quantitatively monitor axonal transport in health and disease.

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals.

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal ...

Unveiling Mitochondrial Ultrastructure in Neurons Using STED Microscopy

Using Live Cell STED Imaging to Visualize Mitochondrial Inner Membrane Ultrastructure in Neuronal Cell Models

Mitochondria play many essential roles in the cell, including energy production, regulation of Ca2+ homeostasis, lipid biosynthesis, and production of reactive oxygen species (ROS). These mitochondria-mediated processes take on specialized roles in neurons, coordinating aerobic metabolism to meet the high energy demands of these cells, modulating Ca2+ signaling, providing lipids for axon growth and regeneration, and tuning ROS production for neuronal development and function. Mitochondrial dysfunction is therefore a central driver in neurodegenerative diseases. Mitochondrial structure and function are inextricably linked. The morphologically complex inner membrane with structural infolds called cristae harbors many molecular systems that perform the signature processes of the mitochondrion. The architectural features of the inner membrane are ultrastructural and therefore, too small to be visualized by traditional diffraction-limited resolved microscopy. Thus, most insights on mitochondrial ultrastructure have come from electron microscopy on fixed samples. However, emerging technologies in super-resolution fluorescence microscopy now provide resolution down to tens of nanometers, allowing visualization of ultrastructural features in live cells. Super-resolution imaging therefore offers an unprecedented ability to directly image fine details of mitochondrial structure, nanoscale protein distributions, and cristae dynamics, providing fundamental new insights that link mitochondria to human health and disease. This protocol presents the use of stimulated emission depletion (STED) super-resolution microscopy to visualize the mitochondrial ultrastructure of live human neuroblastoma cells and primary rat neurons. This procedure is organized into five sections: (1) growth and differentiation of the SH-SY5Y cell line, (2) isolation, plating, and growth of primary rat hippocampal neurons, (3) procedures for staining cells for live STED imaging, (4) procedures for live cell STED experiments using a STED microscope&#160;for reference, and (5) guidance for segmentation and image processing using examples to measure and quantify morphological features of the inner membrane.

Author Spotlight: Decoding Mitochondrial Aging 

This protocol presents a workflow for the propagation, differentiation, and staining of cultured SH-SY5Y cells and primary rat hippocampal neurons for mitochondrial ultrastructure visualization and analysis using stimulated emission depletion (STED) microscopy.

Mitochondria play many essential roles in the cell, including energy production, regulation of Ca2+ homeostasis, lipid biosynthesis, and production of ...

3D Mitochondrial Ultrastructure of Drosophila Indirect Flight Muscle Revealed by Serial-section Electron Tomography

Mitochondria are cellular powerhouses that produce ATP, lipids, and metabolites, as well as regulate calcium homeostasis and cell death. The unique cristae-rich double membrane ultrastructure of this organelle is elegantly arranged to carry out multiple functions by partitioning biomolecules. Mitochondrial ultrastructure is intimately linked with various functions; however, the fine details of these structure-function relationships are only beginning to be described. Here, we demonstrate the application of serial-section electron tomography to elucidate mitochondrial structure in Drosophila indirect flight muscle. Serial-section electron tomography may be adapted to study any cellular structure in three-dimensions.

3D Mitochondrial Ultrastructure of Drosophila Indirect Flight Muscle Revealed by Serial-section Electron Tomography

In this protocol, we demonstrate the application of serial-section electron tomography to elucidate mitochondrial structure in Drosophila indirect flight muscle.

Mitochondria are cellular powerhouses that produce ATP, lipids, and metabolites, as well as regulate calcium homeostasis and cell death. The unique ...

Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

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Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

A Three-Dimensional Technique for the Visualization of Mitochondrial Ultrastructural Changes in Pancreatic Cancer Cells

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Using Live Cell STED Imaging to Visualize Mitochondrial Inner Membrane Ultrastructure in Neuronal Cell Models

3D Mitochondrial Ultrastructure of Drosophila Indirect Flight Muscle Revealed by Serial-section Electron Tomography